The heterogeneous distribution of cholesterol among the membranes of cellular organelles involves processes of transport and sorting that are poorly understood. A large fraction of cholesterol is produced inside lysosomes by hydrolysis of lipoprotein-derived cholesteryl esters. Cholesterol is then released from lysosomes and transferred to other cellular compartments. Our broad goal is to understand the mechanisms of cholesterol export from lysosomes. Lysosomal release of cholesterol is impaired in patients with Niemann Pick disease type C. The faulty gene has been identified and codes for a multispan membrane protein termed NPC1. Immunolocalization studies have suggested that NPC1 shuttles between late endosomes and lysosomes, but the significance of this cycling process and the role of NPC1 in cholesterol transport remain unknown. Two complementary approaches are proposed to address these problems. The intracellular transport of NPC1 and its regulation will be studied using protease protection assays and through analysis of N-linked sugar modifications. In parallel, we will develop novel methodologies, based on scintillation proximity technology, to measure the export of cholesterol from phagolysosomes in macrophages in situ. These experiments are expected to lead to a general understanding of the mechanisms of lysosomal cholesterol release, and will provide the tools to identify other factors involved in this process.